Protein ToxiNs LIFE sTAGEs And Primary Focus

2.1.1 Introduction and Dernitions

Over the past decade, those engaged in the analysis of protein toxins generally focus on one or more attributes of the protein toxin or on the interaction of the toxin with the physiology of the target. Then, applying generally good and sophisticated scientific practice, highly specific conclusions are drawn regarding what was required for a protein to exert toxicity. These highly sophisticated analyses create an impression of the mode of action of individual protein toxins which, if considered in isolation, would lead the reader to believe that protein toxins are each uniquely different and that general rules or general principles are not applicable. From a large collection of articles in this discipline, this review draws together a generalized model for the behavior of protein toxins. Although specific details of any one toxin may be obscured in the process, the overall purpose of this summary is to demonstrate that thinking about protein toxicity, regardless of the protein, can be significantly enhanced by the generalized model presented here.

Foodborne protein toxins are not, in a strict sense, different from protein toxins in general. The principles of protein structure and function leading to toxicity do not differ depending upon the protein being considered. The purpose of this chapter is to articulate a general schematic of how a protein becomes toxic — how it creates a danger to the cells it encounters. The chapter will define what appears today to be the conditions required for all protein toxins to exert toxic effects. The exact "strategy" used by any one protein in creating toxicity differs dramatically among toxins, but the principles of protein toxicity are generally recognized across the entire collection of toxins. In the study of foodborne toxins it is well worth dissecting the general toxin scheme presented here so as to define the toxin strategy and understand any potential threat of a protein agent (familiar or not).

It may be presumptuous to propose that the mode of action of protein toxins might be discussed collectively by using a single diagram, and also that a single set of rules — a single nomenclature — might actually be able to capture the enormous variety of proteins that in one way or another fall into this class. Yet, that is what this chapter will present to describe the biochemical and biophysical principles governing the mode of action — the life cycle of these proteins. In other words, this chapter examines, over a wide range of protein toxins, the general principles and prominent questions being asked by protein toxicologists across a variety of disciplines. Referring to the mode of action within the context of a life cycle creates an impression that the toxic action of a protein is always at risk and the terminal toxic action might be eliminated, reduced, or modulated by a number of events upstream. Similarly, the overall potency of a toxin is determined by upstream events and the specific biophysical interactions governing those events. Figure 2.1 illustrates the general scheme of a protein toxin life cycle that is relevant for all protein toxins. Although each step may be known by several other useful titles, the conventions used here are not arbitrarily chosen but, instead, have been selected to capture the broadest possible application, including applications to the study of foodborne toxins.

The protein toxin life cycle begins with the relative abundance a cell might encounter. This chapter does not focus on the factors contributing to abundance, but essential to the discussion is an appreciation that toxic events depend on a critical abundance of the toxic agent. That abundance must be sufficient to drive all subsequent processes and to cope with any and all factors working against the successful toxic event — factors such as proteolysis, facilitated removal from presentation, inability to activate, achieving the correct solution structure, occlusion from cellular recognition, localization, or critical pretoxic activation. Although not the central focus here, abundance may correct for weakness in downstream processes.

Protein Toxin Lifecycle

Protein Toxin Lifecycle

FIGURE 2.1 Protein toxins can be thought of as having a life cycle where the interval in any one stage of that cycle, illustrated here with an arrow, has implications for the importance of other parts dictated by the specific structure of the protein and ultimately dictating both a "strategy" for killing a cell and the potency of that particular toxin. Each arrow in the diagram can be thought of as part of the strategy.

FIGURE 2.1 Protein toxins can be thought of as having a life cycle where the interval in any one stage of that cycle, illustrated here with an arrow, has implications for the importance of other parts dictated by the specific structure of the protein and ultimately dictating both a "strategy" for killing a cell and the potency of that particular toxin. Each arrow in the diagram can be thought of as part of the strategy.

For example, if the protein toxin is in a highly proteolytic environment, the relative abundance ensures a sufficient amount of toxin to survive to the next steps. If affinities are particularly low, abundance can pick up the slack, ensuring toxicity by driving the on rate in receptor binding.

In some ways it goes without saying that the protein toxin has to be in the right place at the right time — it must be presented to a susceptible tissue. Presentation of the toxin at an active site might be reduced by proteolysis, encapsulation, interaction with denaturants, or blocking peptides or lipids. The rate of movement of the toxin by or through channels where that presentation is likely will modulate the overall presentation, effectively removing the toxic agent from the active site.

It is frequently the case that a protein toxin is in disguise, sometimes protecting the producing organism or sometimes protecting more susceptible sites for later structural alterations. The activation step(s), not to be confused with the final toxin structure required for terminal toxic action, are necessary to maintain abundance and presentation, and may be required for the correct solution structure to mature.

Solution structure refers to the fact that before a toxin binds or is recognized by a cell, with or without activation, the toxin can assume a unique tertiary and quaternary structure determined, in part, by the presenting environment. This solution structure may enhance the lifetime of the toxin. It might even be required for cellular recognition, localization, further alterations, and terminal toxic action.

Although the toxin must be in the right place at the right time (what is referred to here as presentation), cellular recognition is the process that ensures the presence of sufficient abundance for the downstream processes. At this stage the local concentration of the toxin must reach the critical dose. The target cell, functioning with due, honorable intent, may be tricked into admitting what could be a Trojan horse, or double agent. The cell barrier may, on the other hand, keep the toxin close at hand but still capable of a toxic event.

Cellular localization refers to the process of placing the toxin on or in the cell at a place near the target site. In some cases this process is not easily differentiated from cellular recognition. This element in the process may involve more than a single step, such as when a protein toxin must escape from an endocytic vesicle. In this case, the next step of structural alteration might not be easily differentiated from the overall localization process.

The formation of the ultimate toxin structure is the penultimate transition from an inactive entity to a toxic moiety. As mentioned above, this may require additional compartmentalization, but ultimately the toxin assumes a form capable of killing the cell. Although some structural alterations are required for both activation and inac-tivation (usually proteolytic digestion of the protein toxin), the alteration envisioned here is a trigger point that turns a benign protein into a toxin. This step may occur before localization at the site of endpoint toxicity, or may be a modification due to the environment at the endpoint site of toxicity.

The terminal toxic action may be the dramatic destruction of the biological membrane or it may be the subtle hydrolysis of an important regulatory agent. This is frequently the most actively questioned stage in the life cycle. It is this step that many confuse with the entire toxin life cycle, and it is deceptively easy to miscom-municate the terminal toxic event as the mode of action, when by itself the terminal event could not occur without the upstream steps.

Once apoptosis of the cell has begun, proteolysis of cellular macromolecules commences, which may terminate the action of the protein toxin. Some toxins may survive proteolysis and remain fully active and capable of contributing to the relative abundance of the toxin impacting another cell.

Centered in Figure 2.1 is the recurring process of structural alteration, which illustrates that the toxin is created by the interaction of the cell physiology with the protein. At various stages, the primary, secondary, or tertiary structure of the toxin may be altered; in so doing, it may present a new surface or active site capable of increasing the probability that the protein will either be toxic or more readily inactivated and removed from the cell altogether.

Finally, as with all convenient tools, room for confusion is not entirely removed by this protein toxin life cycle. At each stage, the impact of other stages may be recognized and in some cases amplified. The life cycle is merely a convenient way of describing most protein toxins; it creates a common language around which the detailed uniqueness of individual toxins can be recognized and more seriously examined.

2.1.2 Researching a Toxin Life Cycle

In addition to the wide variety of protein toxins under investigation (Table 2.1), researchers will further limit their scope of research based upon the toxin life stage and the central questions under consideration by the discipline. For example, the subject of an investigation may be focused on the signal sequences necessary for cellular localization of a-bungarotoxin. During the past five years, a number of central questions or themes have been identified in the literature, including: proteolytic stability; toxin quaternary structure (toxin-toxin interaction); protein conformational flexibility and triggers; toxin receptor interaction; mechanism of cellular transport and localization; secondary modifications and compartment-dependent conformation; the mechanism of the toxic interaction with the cell; and the elimination of the toxin (inactivation). Table 2.1 is a noninclusive illustration of the overall research on protein toxins defined by the toxin, the life stage, and the central question or theme under investigation.

From this body of research, one can picture the mode of action of any one toxin as a strategy to leverage one or more of the life cycle stages at the point where they dovetail with the target cell physiology. Therefore, the toxicology of any protein toxin is not defined by the life stage per se, as these are common to all protein toxins, though to different degrees. Instead, toxicology is dependent on the debilitating impact of the toxin life stage on a critical cellular event.

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